The group of proteins known as vitamin K-dependent coagulation zymogen proteins, of which Factor VII (FVII) is an example, is of particular interest in the context of the present invention. The proteins in question, which share a similar protein domain structure and have an amino-terminal γ-carboxyglutamic acid (Gla) domain with from 9 to 12 Gla residues, constitute an important class of coagulation factors which are involved in the naturally occurring physiological process known as hemostasis. The process of hemostasis, which occurs in response to bleeding episodes arising as a result of blood-vessel wall damage caused, e.g., by trauma or surgical procedures, is initiated by the formation of a complex between tissue factor (TF), which becomes exposed to the circulating blood following injury to the blood-vessel wall, and an activated form of FVII (FVIIa) which is present in the circulation in an amount corresponding to about 1% of the total FVII protein mass. This complex is anchored to the TF-bearing cell, and it activates Factor X (FX) and Factor IX (FIX) to form activated forms thereof (FXa and FIXa, respectively) on the cell surface. FXa activates prothrombin to thrombin, which in turn activates Factor VIII (FVIII), Factor V (FV), Factor XI (FXI) and Factor XIII (FXIII) to form activated forms thereof (FVIIIa, FVa, FXIa and FXIIIa, respectively).
Furthermore, the limited amount of thrombin formed in this initial step of hemostasis also activates blood platelets by causing them to change shape and expose charged phospholipids on their surface. This activated platelet surface forms a template for subsequent further FX activation and full thrombin generation. The further FX activation on the activated platelet surface occurs via a FIXa-FVIIIa complex formed on the surface of the activated platelet, and FXa then converts prothrombin into thrombin while still on the surface. Thrombin then converts fibrinogen into fibrin, which is insoluble and which stabilizes the initial platelet plug. This process is compartmentalized, i.e. localised to the site of TF expression or exposure, thereby minimizing the risk of a systemic activation of the coagulation system. The insoluble fibrin forming the plug is further stabilised by FXIII-catalyzed cross-linking of the fibrin fibres.
Among the proteins of particular interest in the context of the invention is Factor VII (FVII); FVII proteins occur in mammals (including humans) and numerous other animal genera (e.g. certain fish). FVII exists in plasma mainly as a single-chain zymogen, which is cleaved by FXa into its two-chain activated form, denoted FVIIa. Recombinant activated Factor VIIa (rVFIIa) has been developed as a pro-hemostatic agent. Studies have shown that administration of rFVIIa results in a rapid and highly effective pro-hemostatic response in hemophilic subjects who experience bleeding episodes that cannot be treated with coagulation factor products such as FVIII or FIX owing to antibody formation. Moreover, bleeding subjects with a Factor VII deficiency, as well as subjects having a normal coagulation system but experiencing excessive bleeding (e.g. as a consequence of severe trauma), can be treated successfully with FVIIa. In these studies, no adverse side effects of rFVIIa (in particular the occurrence of thromboembolism) have been encountered.
The administration of extra exogenous FVIIa increases the formation of thrombin on the activated platelet surface; this has been demonstrated with hemophilic subjects lacking FIX or FVIII and therefore lacking the most potent pathway for full thrombin formation. Likewise, in subjects displaying a reduced platelet count or defective platelet function, administration of extra FVIIa increases thrombin formation.
Commercial preparations of recombinant human FVIIa (rhFVIIa) are marketed as NovoSeven™ (Novo Nordisk A/S, Denmark), which is supplied as a freeze-dried formulation in vials containing, e.g., 1.2 mg rhFVIIa, 5.84 mg NaCl, 2.94 mg CaCl2.2 H2O, 2.64 mg glycylglycine (glygly), 0.14 mg Polysorbate™ 80, 60.0 mg mannitol, and which is reconstituted before use using 2.0 ml water for injection (WFI). Once reconstituted, the resulting solution may be used within a period of 24 hours if stored at a maximum of 25° C. No liquid or highly concentrated FVIIa formulations are currently commercially available, but a liquid formulation of FVIIa of adequate activity and stability would clearly be highly desirable.
In general, the stability of a protein in solution may be affected by, inter alia, factors such as ionic strength, pH, temperature, repeated cycles of freeze/thaw, or exposure to shear forces. Active protein may be lost as a result of various kinds of physical or physicochemical instability, including tendency to undergo denaturation and/or aggregation (formation of soluble and/or insoluble aggregates), as well chemical instability, including, for example, tendency to undergo hydrolysis, deamidation, and/or oxidation, to name just a few. For a general review of stability of protein pharmaceuticals, see, for example, Manning et al., Pharmaceutical Research 6: 903-918 (1989).
Whilst the possibility of the occurrence of protein instability is widely recognized, it is largely impossible to make reliable predictions concerning the types of instability problems to be expected for a particular protein. Numerous kinds of instability can result in the formation of a protein by-product, or protein derivative, exhibiting, for example, reduced activity, increased toxicity and/or increased immunogenicity. Thus, for example, in the case of FVIIa, which is a serine protease, fragmentation of the protein due to autoproteolysis is a degradation pathway which has to be taken account of.
Precipitation of protein from solution may at best lead to non-homogeneity of dosage form and amount, as well as to clogging of syringes, or at worst lead to thrombosis in the subject being treated. Furthermore, post-translational modifications such as, for example, gamma-carboxylation of certain glutamic acid residues in the N-terminus of a protein, or the introduction of carbohydrate side-chains, may provide sites that potentially are susceptible to chemical modification upon storage.
Thus, the safety and efficacy of any pharmaceutical formulation of a protein is directly related to its stability. In this respect, maintaining stability in a liquid dosage form is generally more demanding than is the case for a solid preparation, e.g. a lyophilized preparation which is intended for dissolution or reconstitution in an appropriate liquid vehicle shortly before dosing, because of the vastly greater potential for molecular motion—and thereby increased probability of molecular interactions—in the liquid phase. Moreover, maintaining stability in a concentrated liquid formulation of a protein is generally more demanding than is the case with more dilute liquid formulations, because of the greater propensity for aggregate formation at increased protein concentrations.
The present invention arose from the inventors' observation that aqueous liquid preparations/formulations of FVII (in this case as FVIIa) stored in certain types of containers exhibited an unsatisfactorily high rate and/or extent of aggregate formation. On the basis of these observations the present inventors have conducted investigations which have led to measures by which the liquid-phase stability not only of FVII, but also of related types of proteins, may be significantly improved.